SARS‐CoV‐2 Seroprevalence Trends in the Netherlands in the Variant of Concern Era: Input for Future Response

ABSTRACT Background To inform future response planning we aimed to assess SARS‐CoV‐2 trends in infection‐ and/or vaccine‐induced immunity, including breakthrough infections, among (sub)groups, professions and regions in the Dutch population during the Variant of Concern (VOC)‐era. Methods In this prospective population‐based cohort, randomly selected participants (n = 9985) aged 1–92 years (recruited early‐2020) donated home‐collected fingerstick‐blood samples at six timepoints in 2021/2022, covering waves dominated by Alpha, Delta, and multiple Omicron (sub‐)variants. IgG antibody assessment against Spike‐S1 and Nucleoprotein was combined with vaccination‐ and testing data to estimate infection‐induced (inf) and total (infection‐ and vaccination‐induced) seroprevalence. Results Nationwide inf‐seroprevalence rose modestly from 12% (95% CI 11–13) since Alpha to 26% (95% CI 24–28) amidst Delta, while total seroprevalence increased rapidly to 87% (95% CI 85–88), particularly in elderly and those with comorbidities (i.e., vulnerable groups). Interestingly, highest infection rates were noticeable among low/middle educated elderly, non‐Western, those in contact professions, adolescents and young adults, and in low‐vaccination coverage regions. Following Omicron emergence, inf‐seroprevalence elevated sharply to 62% (95% CI 59–65) and further to 86% (95% CI 83–90) in late‐2022, with frequent breakthrough infections and decreasing seroprevalence dissimilarities between most groups. Whereas > 90% of < 60‐year‐olds had been infected at least once, 30% of vaccinated vulnerable individuals had still not acquired hybrid immunity. Conclusions Groups identified to have been infected disproportionally during the acute phase of the pandemic require specific attention in evaluation of control measures and future response planning worldwide. Furthermore, ongoing tailored vaccination efforts and (sero‐)monitoring of vulnerable groups may remain important.


| Introduction
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), causative agent of coronavirus disease 2019 (COVID- 19), has imposed a tremendous burden on societies worldwide since its origin in late 2019.In the Netherlands, stringent measures including social distancing and several lockdowns (with fluctuating stringency; Figure 1A) have been initiated to curb transmission and prevent the health system from collapsing.Nonetheless, initial waves of infection in spring 2020 and later in winter caused huge spikes in hospitalizations and over 20,000 deaths [1].Although some regions were hit relatively hard, only a small proportion of the population had been infected, namely, 2.8% at the peak of the first wave in April 2020 and 4.5% after the first wave in June 2020, as estimated from our earlier nationwide seroepidemiological investigations [2,3].
A nationwide vaccination campaign started in January 2021 and followed an age-, comorbidity-and healthcare worker (HCW)-based prioritization [4].Vaccination coverage was generally high and effectively changed the epidemiology, yet at the same time Variants of Concern (VOC) emerged.The increased transmissibility [5] and severity of Alpha and, thereafter, Delta caused a steep rise in reported cases, hospital-and intensive care unit (ICU) admissions and deaths predominantly among unvaccinated individuals [5][6][7][8][9][10], thus mainly affecting groups with low access and uptake of vaccines [11].All persons aged ≥ 12 years had been offered primary vaccination before the end of 2021.The increasing number of reports of waning vaccine effectiveness triggered administration of booster doses in autumn 2021 [12], (firstly) to vulnerable groups and HCW, and later to everyone ≥ 18 years during winter [4].Signs of enhanced immune escape became even more apparent with emergence of Omicron in late 2021, reflected by high numbers of reported (breakthrough) cases.Relative fewer persons became severely-ill, enabling relaxation of control measures in spring 2022 [10,13].Since the beginning of 2022, primary vaccination has been available for 5-to 11-year-olds, yet nationwide coverage has been low (2%) [10].Second boosters, targeted to at-risk groups, were offered in spring of 2022, and bivalent boosters followed that autumn [14].
To assist public health decision-making during different phases of the pandemic, for example, regarding effectiveness of control measures and vaccination, or insights on severity of disease, it is essential to understand population dynamics of infection and vaccination, and potential disparities between groups [3,8].However, traditional surveillance methods underestimate prevalence and incidence as they rely on testing policy and (self/ home) testing behavior, or because a proportion of cases is asymptomatic and remains undiagnosed [15].Hence, seroepidemiology provides a unique opportunity in complementing other surveillance tools by estimating the prevalence of antibodies induced by infection and/or vaccination, generally indicative of protection against (severe) disease.However, the majority of serosurveys have a number of limitations, for instance: (1) usage of convenience samples that lack representativeness of the general population and in-depth questionnaire data, such as sample sets consisting of only healthcare workers or blood donors, or not including children and elderly; (2) usage of cross-sectional sample sets which carry a risk of missing previous infections due to waning immunity; or (3) sole qualitative assessment of the presence of antibodies, which limits the detection of reinfections [16].
Therefore, by means of the large nationwide population-based prospective PIENTER Corona (PICO) serosurveillance study, we seromonitored the Dutch population repeatedly across all ages throughout the acute phase of the pandemic at multiple timepoints in order to inform (short-term) decision-making.Here, by means of this prospective cohort, we aim to assess trends in infection-and/or vaccine-induced immunity, including breakthrough infections, among different (sub)groups and regions in the population during the VOC-era in 2021-2022.These findings can serve as input for evaluation of the pandemic response and construction of future preparedness globally, and guide ongoing preventive strategies in the endemic/ post-pandemic period.

| Study Settings
Participants were recruited from the Dutch population registry via random selection (without exclusion criteria) in a region-and age-specific (covering 1-90 years) manner.Details on the sampling at the set-up of this prospective PICO-cohort (using the framework of the PIENTER3 serosurvey established in 2016/2017 [17]) and the additional sampling prior to round 2 (PICO2, June 2020, after the first wave) have been described comprehensively [2,3].The PICO-cohort consists of a national sample with participants from across the country resembling the Dutch population (NL); and a sample with participants from municipalities with a lower childhood vaccination coverage (LVC, conceived in PIENTER3), inhabited by a relative large portion of Orthodox-Reformed Protestants who largely refuse vaccination on religious grounds (commonly referred to as "the Bible belt").Prior to PICO6 (November 2021), NL was supplemented similar to previous sampling with randomly-selected persons (predominately younger and older age groups due to drop-outs) to maintain power.Details are provided in the supporting information and Table S1.
The PICO-study protocol was approved by the Medical Ethics Committee MEC-U, the Netherlands (Clinical Trial Registration NTR8473), conformed to the principles embodied in the Declaration of Helsinki, and all participants (or parents/guardians) provided written informed consent.

| Serological Analyses
Serum samples were quantitatively analyzed for SARS-CoV-2 immunoglobulin G (IgG) antibodies against Spike-S1 and Nucleoprotein (N) antigens using a validated fluorescent bead-based multiplex-immunoassay, as described previously [18].Briefly, samples were diluted and incubated with S1-(Sino Biological, 40591-V08H) and N-(Sino Biological, 40588-V08B) coupled beads in SM01-buffer (Surmodics, USA) supplemented with 2% FCS, while shaking at room temperature in the dark for 45 min.Plates were washed three times with PBS, and incubated with PE-conjugated goat anti-human IgG (Jackson ImmunoResearch, 109-116-098) for 30 min.Following final washing steps, samples were acquired on a Luminex FlexMap3D.Antibody concentrations were interpolated from pooled sera calibrated against the World Health Organization (WHO) standard (NIBSC, 20/136) using a 5-parameter logistic fit.Seropositivity for anti-S1 was considered at a cut-off concentration of 10 binding antibody units (BAU) per milliliter (mL) and for anti-N at 14.3 BAU/mL, as derived from previous mixture modeling and receiver operator characteristics analyses [3,19].Applying these cut-offs to detect infection resulted in a specificity for anti-S1 of 99.9% and a sensitivity of 94.3%, and for anti-N of 98.0% and 86.0%, respectively.

| Definitions
At each study round, serological information (since the start of the PICO-study) was combined with vaccination-and testing data to assess infection-induced (inf) (at least once) and total (i.e., infection-and vaccination-induced, equivalent to S1-seropositivity) seroprevalence.More specifically, inf-seroprevalence was determined via anti-S1 seropositivity in unvaccinated (who could potentially serorevert); and in vaccinated individuals via anti-N seropositivity, a SARS-CoV-2 positive test, or a ≥ 4-fold-increase in anti-S1 given they had not received a vaccine dose 4 weeks before their previous collection (i.e., the time for a vaccine response to peak, thereby ruling-out a potential further increase since previous collection due to vaccination and thus fold-increase in the current round).Participants aged ≥ 12 years enrolled since PICO6 were excluded for these infection analyses as they lacked serological history from previous rounds.Since anti-N wanes relatively fast-and was the preferred marker we had to rely on since most were vaccinated at that time-this would have underestimated previous infections (particularly those from the beginning of the pandemic) [19].Moreover, breakthrough infections could be identified in vaccinated persons who presented a positive SARS-CoV-2 test (at least 14 days) after their first vaccination, seroconverted for anti-N in rounds after their first vaccination or had a ≥ 4-fold-increase if already anti-N seropositive, or had a ≥ 4-fold-increase in anti-S1 given they had not received a vaccine dose 4 weeks before their previous collection.

| Analyses
Whole population (overall) and (sub)groups seroprevalence estimates (total and inf), vaccination coverage and -doses, and proportion of breakthrough infections (and their 95% confidence intervals (CIs)) were calculated.The survey design was taking into account and weights (per study sample) were incorporated The PIENTER Corona (PICO) study timeline in 2021 and 2022, by daily occupied hospital beds due to COVID-19 (black line, left y-axis), daily stringency index (gray line, right y-axis) and dominance of specific Variants of Concern (VOC) in the Netherlands (on x-axis, from left to right: Alpha, Delta, Omicron-BA.1, -BA.2, -BA.5, and -BQ.1).A nationwide vaccination campaign started in the beginning of January 2021 and followed an age-, comorbidity-and healthcare worker-based prioritization.The number of participants analyzed in the current study are provided per study round and depicted in italic in the colored boxes.Length of the study rounds are consistent with the width of the colored boxes, although the majority of participants participated at the beginning of each study round, also reflected by the median and interquartile ranges (IQR): PICO4: 17 (15)(16)(17)(18)(19)  The number (proportional to the size of the green dots) and distribution of participants match the countrywide population size and -distribution.Pink-circled municipalities represent the LVC municipalities (as per the PIENTER-3 cohort).Thicker gray lines represent the borders of the provinces and the thin gray lines those of the municipalities.using a set of sociodemographic characteristics (age, sex, ethnic background and urbanization degree) to match the Dutch population distribution following census data (of 1 January 2020) from the Statistics Netherlands.Infection estimates were controlled for test performance characteristics subsequently [20].Differences in seroprevalence between (sub)groups were determined by estimating the parameters of the beta distribution for these estimates using the methods of moments [21].Risk ratios, their corresponding 95% CIs and p values were estimated by Monte Carlo simulations of the seroprevalence estimates; p values were reported where applicable, and those < 0.05 were considered statistically significant.Smooth age-specific seroprevalence estimates were modeled with surveylogistic regressions incorporating B-splines, using Akaike's information criteria (AIC) for optimization of the number of percentile-placed knots.

| Study Population
Table 1 shows the sociodemographic characteristics, comorbidity-and vaccination status of participants throughout the study period.The total number of unique participants with a serological assessment in the current cohort was 9985 and fluctuated between 5767-8549 across study rounds; of which NL comprised 5371-8138 and LVC 275-470.Drop-outs ranged between 8% and 20% per study round, yet the proportions of all sociodemographics groups were comparable over time.In NL, age ranged between 1 and 92 years, with a median between 51-57 years, and generally highest participation in adults up to 80 years.The five sampled regions were consistently similarly (~20%) distributed across the cohort and over time (Figures S1 and 1B).Participants were more often women (56%-57%); of native Dutch origin (88%-89%), as compared to Western (7%-9%) and non-Western (3%-4%); and of lower urbanization degree (49%-50%), versus moderate (31%-32%) or high (19%-20%).Educational level (high vs. middle/low) was nearly equally divided each round.One third could be classified as having comorbidities in 2021, which slightly increased to 4 out of 10 in 2022.The majority of LVC-participants were of native Dutch, low urbanization degree (both > 97%), low/middle educational level (71%-73%), and median age as well as the proportion of having comorbidities (18%-24%) was lower than in NL.
COVID-19 vaccination coverage as well as the number of doses followed a distinct age-pattern over time in NL, consistent with the vaccine roll-out (Figure S2).Overall weighted vaccination coverage (≥ 1 dose) in ≥ 18 years was 5% (95% CI 4-6) in February 2021, rose steeply to 78% (95% CI 76-80) in June, and increased further to 96% (95% CI 95-97) in November 2021.In March 2022, 78% (95% CI 77-80) had received a booster dose, and in June 18% (95% CI 17-20) had been administered a second booster.The latter increased to 43% (95% CI 41-44) in November, while 17% (95% CI [16][17][18][19] had received a fifth dose.Overall coverage did not differ much between sexes, yet was slightly higher in women < 60 years in June 2021 for the primary series, but this difference was nearly erased by the end of the year and coverage remained stable for the doses thereafter.Uptake was consistently lower in the LVC (Table 1).

| Overall, by Age and Region
The overall and stratified, inf-and total seroprevalence are displayed in Figure 2 for 2021 (A) and 2022 (B), and by age in Figure 3 (A: inf; B: total).
In 2022, total seroprevalence already reached 96% (95% CI 95-97) at the beginning of the year and remained high thereafter.
Most infections pre-Alpha were observed in the South-East region (15%) and fewest in the North (10%) (Figure S3 displays all 25 Municipality Health Service [GGD] regions).A fairly similar picture was extended across 2021, with steepest increase in the South-East (31%) and Mid-West (27%), and lowest in the North (20%, p < 0.001 and p = 0.04, respectively).Low urbanized areas had the highest observed inf-seroprevalence throughout the study period, but most pronounced in June 2021 following Alpha (22% vs. high: 17%; p = 0.007).Generally, regional differences did not further intensify in 2022.In LVC, inf-seroprevalence was considerably higher than in NL, especially noticeable till the end of 2021 (~1.5 times: 38% vs. 26%, p = 0.01), and reached > 90% after Omicron-BA.5 at the end of 2022.Correspondingly, the total seroprevalence was nearly consistently lower in that period, illustrative of the lower COVID-19 vaccination coverage.Those who had received a flu vaccination were classified as having underlying comorbidities, consistent with national regulation.NA = not applicable.
Among persons of non-Western descent, inf-seroprevalence rose ~1.5 times sharper during Alpha than in Western and native Dutch, and this was particularly noticeable among < 40-year-olds (Figure S5).Higher inf-seroprevalence persisted among non-Western in all subsequent waves, yet the total seroprevalence was consistently lowest in this group.Moreover, low/middle-educated persons (from age 25 years) had a significantly higher inf-seroprevalence compared to high-educated already pre-Alpha and throughout 2021 (e.g., June: 21% vs. 17%, p = 0.006).This was consistent for all age groups (Figure S6), but most noticeable in individuals aged ≥ 60 years (Alpha: 18% vs. 12%, respectively, p < 0.001; and amidst Delta: 22% vs. 13%, respectively, p = 0.002), coinciding with a lower total seroprevalence in June (p = 0.03).Age group-specific seroprevalence estimates became more comparable after the emergence of Omicron.Further, in persons with underlying comorbidities, targeted for early COVID-19 vaccination, a very high total seroprevalence (> 95%) was already observed in June 2021.Inf-seroprevalence continued to be relatively low in this group compared to those without up until March 2022 after Omicron BA.1 (45% vs. 70%, p < 0.001), yet increased with higher rate thereafter (e.g., after Omicron-BA.5 in November 2022: 73% vs. 91%).

| Occupations
Inf-seroprevalence in the working-age population (18-67 years) followed a similar pattern as the general population throughout 2021-2022.HCW had among the highest inf-seroprevalence pre-Alpha (20%)-together with transportation and production sectors (Figure 4A,B)-which was mainly due to high rates in elderly (home)workers (33%) and hospital employees (22%) (Figure 4C,D).Congruent with a very high total seroprevalence already in June 2021 (> 90%) due to prioritization of vaccination, the rate of infections in HCW slowed down (after Alpha: 26%; amidst Delta: 30%) relative to nationwide, and was similar in March 2022 after Omicron-BA.1.Inf-seroprevalence rates in other HCW sectors were relatively moderate to low until emergence of Omicron-BA.1.Furthermore, sharpest inf-seroprevalence elevations in 2021 were seen among those working in contact professions other than HCW (from 15% in February to 32% in November) and day-care & primary education (from 15% to 29%), and these trends extended into the Omicron waves (e.g., after Omicron-BA.2 [June 2022]: 82% and 91%, respectively; to note, the median age was lowest in these sectors: between 43 and 45 years, vs. others sectors 47-55).Conversely, office employees and those working in middle/higher education had nearly two times lower estimates than HCW after Alpha in June 2021, and only started to catch-up with countrywide estimates thereafter, particularly since emergence of Omicron.

| Discussion
This large Dutch nationwide population-based prospective seroepidemiological study supported public health decisionmaking during the COVID-19 pandemic, for example, as input for the Outbreak Management Team, Ministry of Health, Dutch Health Council, and forecasting/modeling purposes.Our study underscores the importance of serosurveillance globally to supplement other tools in better understanding population immunity and susceptibility over time.The total seroprevalence in NL increased rapidly during the VOC-era, especially in persons prioritized for vaccination.However, infection rates were unequally distributed between (sub)groups and regions pre-Omicron, with highest rates noticeable in adolescents and young adults, low/middle educated elderly, non-Western, those in contact professions including working in childcare, the South-East region and LVC.Following multiple waves of Omicron, the number of breakthrough infections increased steeply, resulting in hybrid immunity for the majority of the Dutch population by the end of 2022.Nevertheless, approximately 3 out of 10 vaccinated vulnerable individuals (i.e., elderly and those with comorbidities) had not acquired hybrid immunity, necessitating tailored vaccination efforts to reduce the risk of severe disease upon infection and ongoing seromonitoring.
Our population-based results are indicative of the prevention of infections by vaccination pre-Omicron and in line with vaccine-effectiveness studies as well as seroepidemiological studies conducted in regions with high vaccination coverage FIGURE 2 | (Continued) [12].Particular illustrative was the relative modest increase in infections in HCW since vaccination despite having the highest exposure-potentially in combination with adequate use of personal protective equipment.Conversely, the rate of infection remained high in the South-Eastern part of the country, that is, the epicenter of the first wave in 2020, and particularly in LVC [2].A combination of lower vaccine uptake (consistent with lower total seroprevalence) and increased exposure, for example, due to lower adherence to measures, might have caused this and has also been observed in similar communities abroad, such as the United Kingdom [4,22].Future research should gain more understanding of the concerns in harder-to-reach regions and apply this knowledge as input for tailor-made preventive programs.Further, in-depth research on air quality across regions and severity of COVID-19 has shown an increased risk in the lower urbanized areas in the South-East, recognized for poorer air quality due to intensive livestock farming [23].
Importantly, relatively higher rates of infection were also observed in low/middle educated elderly and non-Western, particularly after roll-out of vaccination.The pandemic entails a double burden for the most disadvantaged groups: increased likelihood of infection, for example, due to fewer opportunities to work remotely, as well as severe disease due to weaker health or access to health.Our data are congruent with lower vaccination uptake and higher ICU admissions in these specific groups in our country and abroad during this period, such as other to working remotely.Also, despite the highest rates in young adults, professions in middle/higher education were comparable to nationwide estimates.However, unless demonstrative beneficial effects of limited contact and remote schooling on reducing viral transmission, potential detrimental social-and cognitive effects should be taken into account while evaluating the pandemic response [28].
To our knowledge this is the first study to present population estimates of breakthrough infections across various VOCs.
After the first Omicron wave and relaxation of control measures, the rate of infections had increased steeply across the population, consistent with reports globally [16], and differences in seroprevalence between groups shrunk.A large elevation in the proportion of breakthrough infections was observed, firstly among adolescents up until middle-aged adults and in later waves across all adults.This trend is most likely explained by increased booster doses in elderly, which have shown to be effective against infection in the short term, but susceptibility increased due to waning immunity alongside novel variants with enhanced immune-escape features [12].Hybrid immunity has proven to be most effective against future infection [29], potentially mediated by mucosal antibodies in the upper respiratory tract [30,31], and severe disease [32].These observations on susceptibility in the most vulnerable persons contributed to targeted prevention strategies with novel booster doses for winter 2023, and warrants ongoing seromonitoring (of vaccine-effectiveness).
This study has several strengths and limitations that should be highlighted.This is one of the largest prospective populationbased serological cohorts, including children and a unique LVC sample, that was set-up since the beginning of the pandemic and is still ongoing.Since (self-)testing has been reduced significantly after relaxation of control measures in 2022, serological assessment including boosting of antibodies, is essential for detection of (breakthrough) infections, and for guiding surveillance.Anti-N generally has a somewhat lower sensitivity than anti-S1 and is known to wane quicker [19,33] which might have caused some underestimation of breakthrough infections.However, we did not observe a significant reduction in sensitivity of anti-N in vaccinees fortunately [34], which has been reported by some [35], and due to our repeated sampling with short intervals we still expect high case ascertainment.Moreover, in-depth questionnaire data allowed investigation into (sub)groups.Response rates in children were rather low and drop-out rates high, however since children were less restricted by control measures and schools were mostly open, we do not expect large differences between children in terms of exposure, and this was also confirmed by contact data [36,37].Despite random selection and weighting our sample, some groups are underrepresented, such as those living in nursing homes-that were hit hard pre-Alpha-and non-Western, who might have refrained from participation due to digital-and/or language barriers.Study participants may generally adhere better to control measures, also reflected by a higher vaccination coverage than the general population, which could have underestimated inf-seroprevalence especially pre-Omicron.
To conclude, this seroepidemiological study has provided important insights on dynamics of SARS-CoV-2 population immunity resulting from (breakthrough) infections and vaccination during the VOC-era.These results will be key in evaluation of control measures, construction of future pandemic response planning, and guiding ongoing preventive strategies for the most vulnerable groups.In the endemic/post-pandemic phase, this robust serological framework will be vital for the relevant challenges ahead, such as unraveling risk factors for post-COVID and mucosal immunity, but also in gaining knowledge on the (potentially permanent) epidemiological changes observed for other respiratory pathogens during the pandemic, for example, respiratory syncytial virus and group A streptococcal [38,39].
FIGURE 1 | (A)The PIENTER Corona (PICO) study timeline in 2021 and 2022, by daily occupied hospital beds due to COVID-19 (black line, left y-axis), daily stringency index (gray line, right y-axis) and dominance of specific Variants of Concern (VOC) in the Netherlands (on x-axis, from left to right: Alpha, Delta, Omicron-BA.1, -BA.2, -BA.5, and -BQ.1).A nationwide vaccination campaign started in the beginning of January 2021 and followed an age-, comorbidity-and healthcare worker-based prioritization.The number of participants analyzed in the current study are provided per study round and depicted in italic in the colored boxes.Length of the study rounds are consistent with the width of the colored boxes, although the majority of participants participated at the beginning of each study round, also reflected by the median and interquartile ranges (IQR): PICO4: 17(15)(16)(17)(18)(19) February 2021; PICO5: 23 (21-28) June 2021; PICO6: 11 (8-15) November 2021; PICO7: 23 (19-28) March 2022; PICO8: 21 (19-27) June 2022; and PICO9: 9 (5-14) November 2022.Data on hospital bed occupation are open source and were downloaded from the National Coordination Center for Patient Distribution (LCPS) (via: https:// lcps.nu/ datafeed).Stringency index data, here depicted for the Netherlands, are open source too and were downloaded from Our World in Data (via: https:// ourwo rldin data.org/ covid -strin gency -index .(B) Nationwide distribution of the total number of unique PICO participants analyzed (n = 9985), by municipality.The current study comprises the national sample (n = 9492) and the low vaccination coverage (LVC) sample (n = 493).The number (proportional to the size of the green dots) and distribution of participants match the countrywide population size and -distribution.Pink-circled municipalities represent the LVC municipalities (as per the PIENTER-3 cohort).Thicker gray lines represent the borders of the provinces and the thin gray lines those of the municipalities.
Numbers (n) and proportion (%) were provided for participants in the current PIENTER Corona (PICO) study cohort belonging to the national sample and low vaccination coverage (LVC) sample, covering study rounds in 2021 (February [PICO4], June [PICO5] and November [PICO6]) and 2022 (March [PICO7], June [PICO8], November [PICO9]), by sociodemographic characteristics, and comorbidity-and vaccination status.Educational level (from 25 years of age, highest obtained or current) was classified as low (no education or primary education)/middle (secondary school or vocational training), or high (bachelor's degree, university).Regions consist of the following provinces: North = Groningen, Friesland, Drenthe and Overijssel; Mid-West = Flevoland and Noord-Holland; Mid-East = Gelderland and Utrecht; South-West = Zuid-Holland and Zeeland; South-East = Noord-Brabant and Limburg.

TABLE 1 |
Description of the PIENTER Corona (PICO) study cohort in 2021 and 2022, covering study rounds 4 to 9.